The invention relates to the genetic manipulation of plants, particularly to the expression of hydroperoxide lyase (HPL) genes in transformed plants.
Plants produce volatile aldehydes via the oxylipin pathway; first converting a fatty acid to a hydroperoxy fatty acid by the action of a lipoxygenase, and subsequently cleaving the hydroperoxy fatty acid into a volatile aldehyde and an aldehyde acid by the action of a hydroperoxide lyase (HPL). For example, via the oxylipin pathway, linoleic acid converted to its hydroperoxy form is subsequently cleaved by HPL to yield the volatile 6 carbon aldehyde N-hexanal and 12-oxo-9Z-dodecenoic acid.
Similarly, linolenic acid converted to its hydroperoxy form is cleaved by HPL to yield 3Z-hexenal and 12-oxo-9Z-dodecenoic acid. 3Z-hexenal is further converted to 2E-Hexenal (leaf aldehyde) by an isomerase factor. HPL also catalyzes formation of 9-carbon aldehydes such as nonadienal and the corresponding nonanoic acid. Action of an alcohol dehydrogenase converts the oxylipin pathway-derived aldehydes to their corresponding alcohols; for example hexanol, hexenol or nonadienol. A review of volatile aldehyde formation in plants is provided in Hatanaka et al (1986) Biogeneration of Aromas, American Chemical Society, 167-175, the contents of which are herein incorporated by reference.
Food toxicoses affect human and animal health worldwide. Aflatoxin contamination has been identified as a result of fungal infection with the Aspergillus flavus (A. flavus) group in many foods, including corn grown in Southeastern United States. Lack of aflatoxin contamination in early stages of corn development is associated with presence of volatile metabolites at peak concentrations. Naturally occurring volatile aldehydes inhibit A. flavus growth in corn (Gueldner et al. (1985) J. Agric. Food Chem. 33(3):411-413); and the exceptional natural resistance of soybeans to A. flavus may be attributed to generation of volatile aldehydes, particularly hexanal (Doehlert et al. (1993) Abstract, American Phytophathological Society 83(12):1473-1477). A relationship between aflatoxin resistance, and volatile aldehyde, and precursor fatty acid content, has been reported in studies of aflatoxin-susceptible and -resistant maize (Zeringue, Jr. et al. (1996) J. Agric. Food Chem. 44:403-407). However, a concentration-dependent toxicity to higher plants has also been shown to be associated with volatile aldehydes (Gardner et al. (1990) J. Agric. Food Chem. 38:1316-1320).
Volatile aldehydes can inhibit growth of fungal cultures of Colletotrichum truncatum, Rhizoctonia solani and Sclerotium rolfsii (Vaughn et al. (1993) Journal of Chemical Ecology 19(10):2337-2345). Furthermore, volatile aldehydes can exhibit antibacterial activity and are implicated in protection against mechanical wounding; for example, insect-induced wounding (Matsui et al. (1996) FEBS Letters 394:21-24; Shibata et al (1995) Plant Cell Physiol. 36(1):147-156; Blxc3xa9e et al. (1996) Plant Physiol. 110:445-154).
Six carbon aldehydes, together with their corresponding alcohols are responsible for the characteristic odor of green leaves, and are also constituents of aroma and flavor from various fruits. As such aroma and flavor compounds, the aldehydes are also referred to as natural xe2x80x9cgreen notexe2x80x9d compounds (Hatanaka et al. (1986) Biogeneration of Aromas, American Chemical Society, 167-175; Gxc3x6tz-Schmidt et al. (1986) Lebensm.-Wiss. u.-Technol. 19(2):152-155; EP 0 801 133 A2; Matsui et al. (1996) FEBS Letters 394:21-24).
HPL is distributed in a variety of plant species in membrane bound forms; both chloroplastic and non-chloroplastic. HPL has been purified from both non-green and green tissue; including fruits of pear, tomato, cucumber, cultured cells of tobacco, and tea leaves (Hatanaka et al. (1986) Biogeneration of Aromas, American Chemical Society, 167-175; Gxc3x6tz-Schmidt et al. (1986) Lebensm.-Wiss. u.-Technol 19(2):152-155). It has been suggested that two forms of HPL exist; one is common to both green and non-green cells and another is chloroplast-specific (Sekiya et al. (1984) Phytochemistry 23(11):2439-2443). HPL-like activity has also been observed in non-plant microbial sources (Bisakowski et al. (1997) Biosci. Biotech. Biochem. 61(8):1262-1269).
While the biochemistry and tissue distribution of HPL is well characterized, data regarding molecular biology of HPL is sparse. HPL has been cloned from bell pepper. Sequence analysis of the clone shows C-terminal homology to members of the cytochrome P450 family, particularly to allene oxide synthase. Heme- and oxygen-binding domains have been identified in this sequence (Matsui et al. (1996) FEBS Letters 394:21-244). HPL has also been cloned from banana leaf as described in EP 0 801 133 A2, and from Arabidopsis as described in Bate et al. (1998) Plant Physiol 117(4):1393-1400; although no DNA sequence is described for the Arabidopsis HPL clone.
Due to the role of volatile aldehydes in disease resistance, pathogen protection, and modulation of aroma and flavor, it would be beneficial to influence levels of volatile aldehyde and related compounds by manipulating HPL levels in a plant.
Compositions and methods for expressing hydroperoxide lyase (HPL) genes in plants, plant cells, and plant tissues are provided. The compositions comprise nucleotide sequences encoding maize HPL genes. The sequences are useful in transforming plants for tissue-preferred or constitutive expression of HPL. Such sequences find use in enhancing disease resistance and in modulating levels of flavor molecules in plants.
Expression cassettes comprising the HPL sequences of the invention are provided. Additionally provided are transformed plant cells, plant tissues, and plants.